Description:

Workflow begin

This tutorial comes from Seurat

Load required packages

library(dplyr)
library(Seurat)
library(leiden)
library(ggplot2)
library(patchwork)

Load data

Input is a directory that contains CellRanger count outputs

previous_time <- proc.time()[3]
Estrus.data <-Read10X(data.dir ="/home/nguyen/Estrus_cellranger_count/Estrus_Raw/")
proc.time()[3] - previous_time
elapsed 
  7.678 
  • View(Estrus.data) - run this command to investigate the output
  • Current dataset:
    • 31253 genes
    • 6599 cells

Initialize the Seurat object

  • Initialize the Seurat object with the raw (non-normalized data).
  • Filter out genes that are detected in less than 3 cells
  • Filter out cells that have less than 200 genes expressed
previous_time <- proc.time()[3]
Estrus <- CreateSeuratObject(counts = Estrus.data, project = "Estrus", min.cells = 3, min.features = 200)
proc.time()[3] - previous_time
elapsed 
   1.49 
Estrus
An object of class Seurat 
19420 features across 6481 samples within 1 assay 
Active assay: RNA (19420 features, 0 variable features)
  • Output:
    • 19420 genes
    • 6481 cells

Mitochondiral genes overview

# This is the list of genes that start with mt
mito_genes <- grep("^mt-", rownames(Estrus) , ignore.case=T, value=T)
print(mito_genes)
 [1] "mt-Nd1"  "mt-Nd2"  "mt-Co1"  "mt-Co2"  "mt-Atp8" "mt-Atp6" "mt-Co3"  "mt-Nd3"  "mt-Nd4l" "mt-Nd4"  "mt-Nd5"  "mt-Nd6"  "mt-Cytb"

mt-Nd6 and mt-Cytb will be manually deleted

counts <- GetAssayData(Estrus, assay = "RNA")
counts <- counts[-(which(rownames(counts) %in% c("mt-Nd6", "mt-Cytb"))),]
Estrus <- subset(Estrus, features = rownames(counts))
# This is the list of genes that start with mt
mito_genes <- grep("^mt-", rownames(Estrus) , ignore.case=T, value=T)
print(mito_genes)
 [1] "mt-Nd1"  "mt-Nd2"  "mt-Co1"  "mt-Co2"  "mt-Atp8" "mt-Atp6" "mt-Co3"  "mt-Nd3"  "mt-Nd4l" "mt-Nd4"  "mt-Nd5" 
  • “mt-Nd6”, “mt-Cytb” were deleted

Calculate the percentage of mitochondrial genes

  • Add “percent.mt” column to object metadata
  • Percentage of mitochondrial genes is stored in “percent.mt”
  • “^mt-” means strings start with mt (lowercase mt for mice, uppercase MT for human)
Estrus[["percent.mt"]] <- PercentageFeatureSet(Estrus, pattern = "^mt-")
# This is the data in "percent.mt" column
percent_mito <- FetchData(object = Estrus, vars = "percent.mt") #how to fetch data
head(percent_mito)
# Visualize QC metrics as a violin plot
VlnPlot(Estrus, features = c("nFeature_RNA", "nCount_RNA", "percent.mt"), ncol = 3)
# FeatureScatter is typically used to visualize feature-feature relationships, but can be used
# for anything calculated by the object, i.e. columns in object metadata, PC scores etc.
plot1 <- FeatureScatter(Estrus, feature1 = "nCount_RNA", feature2 = "percent.mt")
plot2 <- FeatureScatter(Estrus, feature1 = "nCount_RNA", feature2 = "nFeature_RNA")
plot1 + plot2

Filter out low-quality cells

  • We filter out cells that have unique feature counts over 6000
  • Because any cell that has more than 6000 genes are likely doublets
  • Note: Seurat tutorial used 2500 instead of 6000
  • We also filter out cells that have more than 5% mitochondrial genes
  • Because those are likely to be nonviable cells
previous_time <- proc.time()[3]
Estrus <- subset(Estrus, subset = nFeature_RNA < 6000 & percent.mt < 5)
proc.time()[3] - previous_time
elapsed 
  0.327 
Estrus
An object of class Seurat 
19418 features across 2585 samples within 1 assay 
Active assay: RNA (19418 features, 5000 variable features)
 1 dimensional reduction calculated: pca
  • After filtering step, the dataset is left with
  • 19418 genes
  • 2585 cells

Normalizing the data

  • Perform log-normalization method
  • Feature counts for each cell are divided by the total counts for that cell and multiplied by the scale.factor
  • scale.factor default value = 10,000
  • This is then natural-log transformed using log1p
previous_time <- proc.time()[3]
Estrus <- NormalizeData(Estrus, normalization.method = "LogNormalize", scale.factor = 10000)
Performing log-normalization
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
proc.time()[3] - previous_time
elapsed 
  0.261 

Identification of highly variable features (feature selection)

previous_time <- proc.time()[3]
Estrus <- FindVariableFeatures(Estrus, selection.method = "disp", nfeatures = 5000)
Calculating gene means
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variance to mean ratios
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
proc.time()[3] - previous_time
elapsed 
  0.493 

Plotting highly variable features

# Identify the 10 most highly variable genes
top10 <- head(VariableFeatures(Estrus), 10)
top10
 [1] "Hbb-bs" "Hba-a1" "Hba-a2" "Hbb-bt" "Dcpp3"  "Ccl21a" "Apoe"   "Cd74"   "Ccl5"   "Dcpp1" 
# Plot variable features with and without labels
plot1 <- VariableFeaturePlot(Estrus)
plot2 <- LabelPoints(plot = plot1, points = top10, repel = TRUE)
When using repel, set xnudge and ynudge to 0 for optimal results
plot1 + plot2 
Warning: Removed 1 rows containing missing values (geom_point).
Warning: Removed 1 rows containing missing values (geom_point).

Regress out data and perform scaling

  • Regressing out total counts per cell and percent.mt before scaling.
  • Take out mitochondrial genes before scaling (analyzing) the data
  • Because mito_genes shouldn’t be accounted for cell population analysis.
previous_time <- proc.time()[3]
all.genes <- rownames(Estrus)
Estrus <- ScaleData(Estrus, features = all.genes, vars.to.regress = c("nCount_RNA","percent.mt"), scale.max = 10)
Regressing out nCount_RNA, percent.mt

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Centering and scaling data matrix

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# Explanations below are from [Seurat](https://satijalab.org/seurat/archive/v3.0/cell_cycle_vignette.html)
# vars.to.regress = RegressOut in Seurat version 1.4 or lower
# However, as the results of this procedure are stored in the scaled data slot (therefore overwriting the output of ScaleData) 
# This functionality is merged into the ScaleData function itself.
proc.time()[3] - previous_time
elapsed 
724.018 

Perform linear dimensional reduction (PCA)

previous_time <- proc.time()[3]
Estrus <- RunPCA(Estrus, features = VariableFeatures(object = Estrus))
PC_ 1 
Positive:  Rps28, Rps19, Rpl18a, Rpl13, Rpl23a, Rplp0, Rpl37, Rpl32, Rps12, Rps18 
       Rps5, Rps24, Rps29, Rpl37a, Rps7, Rps27a, Rps23, Rps11, Rpl35, Rps4x 
       Rpl39, Cd63, Rps20, Rpl27a, Rps15a, Rps16, Rpl10, Rps3a1, Rpl19, Rps9 
Negative:  Gm867, 1700016K19Rik, Ccdc153, 1700007K13Rik, Tmem212, Dynlrb2, Gm19935, 1110017D15Rik, Cfap126, Fam166c 
       Fam183b, Sntn, Vpreb3, Foxj1, Rsph1, Tctex1d4, Dnali1, Lrrc10b, Pifo, 1700024G13Rik 
       Prr29, Dnah12, Hdc, Mlf1, 2410004P03Rik, Tppp3, Efcab10, Timp4, Dnah5, Elof1 
PC_ 2 
Positive:  Lcn2, Cd68, Fxyd3, Arg1, Sftpd, Wfdc2, Sprr2f, Pdzk1ip1, Ltf, Prap1 
       Cxcl17, Serpina1e, Clca1, Padi1, Pigr, Rnf128, Muc1, Tmem45b, Padi2, Muc4 
       Ly6a, Tacstd2, Epcam, Ckmt1, Fxyd4, Elf3, Trpv6, Pglyrp1, Car2, Fut9 
Negative:  Serpinh1, Sparc, Col1a2, Col3a1, Cald1, Col1a1, Col6a1, Bgn, Mgp, Igfbp7 
       Col6a2, Rarres2, Aebp1, Zbtb20, Dcn, Nbl1, Rcn3, Igfbp6, Mdk, Sparcl1 
       Csrp2, Fxyd1, Mfap2, Fxyd6, Col6a3, Lgals1, Col4a1, Igf1, Pbx1, Gsn 
PC_ 3 
Positive:  Ovgp1, Gstm2, Wfdc18, Emb, Kctd14, Plet1, Aldoc, Krt18, Tpd52l1, Clu 
       Agr2, Cldn22, Gstm1, Tmem218, Cbs, Ppp1r1b, Hddc2, Slc1a3, Rcn1, Lrpap1 
       Spint2, Shisa8, Dkk3, Rbp1, Kcne3, Manf, Tmed3, Dcxr, Mgst1, Cystm1 
Negative:  Ctss, Tyrobp, Fcer1g, Csf1r, Laptm5, Apoe, Cd52, H2-Ab1, Cd74, H2-Aa 
       H2-Eb1, Coro1a, C1qc, Aif1, C1qa, C1qb, Lyz2, H2-DMa, Cx3cr1, Pld4 
       Ms4a6c, Cybb, Ly86, Ms4a7, Spi1, Mpeg1, B2m, Trf, Fyb, Fcgr3 
PC_ 4 
Positive:  Clca1, Cxcl17, Muc4, Prap1, Pigr, Serpina1e, Slc40a1, Plac8, Ly6a, Sprr2f 
       Tmem45b, Trpv6, Arg1, Tacstd2, Tmprss4, Fxyd4, Prxl2a, Padi2, Tspan1, Fut9 
       Fcgbp, Ckmt1, Car2, Lrg1, Morrbid, Pdzk1ip1, Wfdc2, Upk3bl, Sftpd, Ncoa7 
Negative:  Ctsc, Tyrobp, Fcer1g, Cd52, H2-Aa, C1qc, Cd74, Csf1r, Aif1, C1qa 
       C1qb, H2-Eb1, Lyz2, Coro1a, H2-Ab1, Cx3cr1, Pld4, Ly86, Ms4a6c, Cybb 
       Ms4a7, Spi1, Cyth4, Fcgr3, H2-DMa, Lcp1, Mpeg1, Laptm5, Lst1, Adgre1 
PC_ 5 
Positive:  Gsn, Pdgfra, Htra3, Mfap4, Mfap2, Dpt, Serpinf1, Sparcl1, Ntrk2, Col6a3 
       Col15a1, Penk, Gpm6b, Lama2, Lpl, Matn2, Ramp3, Mdk, Spon1, Ramp1 
       Rxfp1, Col5a2, Islr, Osr2, Srpx, Itih5, Zeb2, Htra1, Bgn, Enpp2 
Negative:  Cavin2, Gpm6a, Kdr, Cav1, Egfl7, Podxl, Ecscr, Adgrf5, Cldn5, Pecam1 
       Esam, Cdh5, Prss23, Arhgap29, Upk3b, Mmrn1, Tie1, Flt4, Lrrn4, Upk1b 
       Nkain4, Emcn, Stmn2, Ccl21a, C2, Ltbp4, Ushbp1, Mmrn2, Myrf, Lmcd1 
proc.time()[3] - previous_time
elapsed 
  4.545 

Examine and visualize PCA results a few different ways

print(Estrus[["pca"]], dims = 1:5, nfeatures = 5)
PC_ 1 
Positive:  Rps28, Rps19, Rpl18a, Rpl13, Rpl23a 
Negative:  Gm867, 1700016K19Rik, Ccdc153, 1700007K13Rik, Tmem212 
PC_ 2 
Positive:  Lcn2, Cd68, Fxyd3, Arg1, Sftpd 
Negative:  Serpinh1, Sparc, Col1a2, Col3a1, Cald1 
PC_ 3 
Positive:  Ovgp1, Gstm2, Wfdc18, Emb, Kctd14 
Negative:  Ctss, Tyrobp, Fcer1g, Csf1r, Laptm5 
PC_ 4 
Positive:  Clca1, Cxcl17, Muc4, Prap1, Pigr 
Negative:  Ctsc, Tyrobp, Fcer1g, Cd52, H2-Aa 
PC_ 5 
Positive:  Gsn, Pdgfra, Htra3, Mfap4, Mfap2 
Negative:  Cavin2, Gpm6a, Kdr, Cav1, Egfl7 
VizDimLoadings(Estrus, dims = 1:2, reduction = "pca")

DimPlot(Estrus, reduction = "pca") + ggtitle(label = 'Including immune cells') 
ggsave(filename = "PCA_Including_immune_cells.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 7.29 x 4.5 in image

FeaturePlot(Estrus, reduction = "pca", features = c('Ovgp1', 
                                                    'Foxj1', 'Igf1', 'Pdxk', 
                                                    'Fxyd4', 'Bsg', 
                                                    'Serpina1e', 'Krt19'))

ggsave(filename = "PCA_Including_immune_cells1.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image
FeaturePlot(Estrus, reduction = "pca", features = c('Dcn', 'Pax8',
                                                    'Ephx2', 
                                                    'Pecam1', 'Pdgfra', 'Myh11', 'Atp2b4', 
                                                    'Cd52'))

ggsave(filename = "PCA_Including_immune_cells2.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image

Determine the ‘dimensionality’ of the data

  • Seurat tutorial uses JackStraw() and and ScoreJackStraw to determine dimensionality
  • the syntax is:
    • Estrus <- JackStraw(Estrus, num.replicate = 100)
    • Estrus <- ScoreJackStraw(Estrus, dims = 1:20)
  • However, it takes very long.
  • An elbow plot often corresponds well enough with the significant dims and is much faster to run than Jackstraw
  • According to the graph below, choose the first 22 principle components to use
ElbowPlot(Estrus, ndims = 40)

Find neighbors

  • Dims = dimensions = determined as 22 from the above step
previous_time <- proc.time()[3]
Estrus_new <- FindNeighbors(Estrus, dims = 1:22, k.param = 10)
Computing nearest neighbor graph
Computing SNN
proc.time()[3] - previous_time
elapsed 
  0.324 

Find cell clusters using Leiden’s method

# Algorithm = 4 specifies Leiden's method
previous_time <- proc.time()[3]
Estrus_new <- FindClusters(Estrus_new, resolution = 0.14, algorithm = 4)
proc.time()[3] - previous_time
elapsed 
  0.754 

Constructing UMAP plot

previous_time <- proc.time()[3]
Estrus_new <- RunUMAP(Estrus_new, dim = 1:22) 
16:23:56 UMAP embedding parameters a = 0.9922 b = 1.112
16:23:56 Read 2585 rows and found 22 numeric columns
16:23:56 Using Annoy for neighbor search, n_neighbors = 30
16:23:56 Building Annoy index with metric = cosine, n_trees = 50
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
16:23:57 Writing NN index file to temp file /tmp/RtmprVQ6Uz/file19849734bf80
16:23:57 Searching Annoy index using 1 thread, search_k = 3000
16:23:57 Annoy recall = 100%
16:23:57 Commencing smooth kNN distance calibration using 1 thread
16:23:58 Initializing from normalized Laplacian + noise
16:23:58 Commencing optimization for 500 epochs, with 102266 positive edges
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
16:24:00 Optimization finished
proc.time()[3] - previous_time
elapsed 
  3.653 

Visualization

DimPlot(Estrus_new, reduction = "umap") + ggtitle(label = 'Including immune cells')
ggsave(filename = "UMAP_Including_immune_cells.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 7.29 x 4.5 in image

FeaturePlot(Estrus_new, reduction = "umap", features = c('Ovgp1', 'Foxj1', 'Dcn', 'Serpina1e', 'Sprr2f',
                                                         'Fxyd4', 'Ly6a', 'Epcam', 'Krt19', 'Vim', 'Pdgfra'))

ggsave(filename = "UMAP_Including_immune_cells1.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image
FeaturePlot(Estrus_new, reduction = "umap", features = c('Myh11', 
                                        'Dkk2', 'Slitrk6', 'Dio2', 'Smoc2', 'Tes', 'Dab1', 'Camk1d', 'Pecam1', 'Cd52','Mki67'))

ggsave(filename = "UMAP_Including_immune_cells2.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image

Removing immune cells

sub_Estrus = subset(x = Estrus_new, ident = c(1,2,3,4,5,6,7,9,10,12))
DimPlot(sub_Estrus, reduction = "umap") + ggtitle(label = 'Immune cells removed')

ggsave(filename = "UMAP_Immune_cells_removed.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image
FeaturePlot(sub_Estrus, reduction = "umap", features = c('Ovgp1', 'Foxj1', 'Fxyd4', 'Cxcl17', 'Myh11', 'Cd52', 'Twist2', 'Pecam1'))

ggsave(filename = "UMAP_Immune_cells_removed1.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image
FeaturePlot(sub_Estrus, reduction = "umap", features = c('Pdgfra', 'Ephx2', 'Dcn', 'Krt19', 'Epcam', 'Vim','Mki67'))

ggsave(filename = "UMAP_Immune_cells_removed2.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image
sub_Estrus
An object of class Seurat 
19418 features across 2318 samples within 1 assay 
Active assay: RNA (19418 features, 5000 variable features)
 2 dimensional reductions calculated: pca, umap
  • After removing immune cells, the dataset is left with:
    • 19418 genes
    • 2318 cells

Re-run analysis after excluding immune cells

previous_time <- proc.time()[3]
sub_Estrus <- RunPCA(sub_Estrus, features = VariableFeatures(object = sub_Estrus))
PC_ 1 
Positive:  Gm867, Ccdc153, 1700016K19Rik, 1700007K13Rik, Tmem212, Fam166c, Dynlrb2, Gm19935, Sntn, 1110017D15Rik 
       Fam183b, Cfap126, Foxj1, Vpreb3, Dnali1, Rsph1, Tctex1d4, Lrrc10b, 1700024G13Rik, Dnah12 
       Pifo, Prr29, 2410004P03Rik, Mlf1, Hdc, Tppp3, Dnah5, Efcab10, Timp4, Dnaja4 
Negative:  Rps28, Rps19, Rplp0, Rpl18a, Rpl13, Rpl23a, Rpl37, Rpl32, Rps18, Rps5 
       Rps7, Rps12, Rpl37a, Rps24, Rps27a, Cd63, Rpl39, Rps23, Rps4x, Rps29 
       Rps20, Rpl35, Rpl27a, Rps15a, Rps11, Rps3a1, Rpl10a, Rpl10, Rps2, Rps16 
PC_ 2 
Positive:  Cald1, Col1a2, Serpinh1, Col3a1, Col6a1, Col1a1, Aebp1, Bgn, Rarres2, Mgp 
       Sparc, Col6a2, Dcn, Igfbp7, Igfbp6, Rcn3, Rnase4, Zbtb20, Mdk, Lgals1 
       Nbl1, Csrp2, Fxyd1, Igf1, Sparcl1, Lrp1, Mfap2, Col6a3, Timp2, Ccdc80 
Negative:  Lcn2, Fxyd3, Ltf, Epcam, Sftpd, Wfdc2, Plet1, Arg1, Pdzk1ip1, Rnf128 
       Sprr2f, Muc1, Padi1, Prap1, Cxcl17, Cldn7, Elf3, Serpina1e, Clca1, Ier3 
       Pigr, Ly6a, Ckmt1, Tmem45b, Car2, Muc4, Bcat1, Tspan8, Pglyrp1, Tacstd2 
PC_ 3 
Positive:  Ovgp1, Kctd14, Gstm2, Emb, Clu, Tmem218, Gstm1, Cbs, Wfdc18, Ppp1r1b 
       Slc1a3, Aldoc, Agr2, Cldn22, Dkk3, Rcn1, Lrpap1, Shisa8, Fn1, Tpd52l1 
       Sdf2l1, Smim14, Selenom, Manf, Gdpd2, Bex4, Enho, Tmed3, Kcne3, Slco2a1 
Negative:  Plac8, Clca1, Muc4, Pigr, Cxcl17, Ly6a, Slc40a1, Prap1, Trpv6, Tmem45b 
       Serpina1e, Sprr2f, Tspan1, Tmprss4, Tacstd2, Padi2, Prxl2a, Fcgbp, Cfh, Arg1 
       Fxyd4, Fut9, Lrg1, Rasa4, Fbln1, Morrbid, Slc39a8, Ckmt1, Ncoa7, Upk3bl 
PC_ 4 
Positive:  Upk3b, Nkain4, Lrrn4, Upk1b, C2, Myrf, Tmem151a, Lgals7, Fgf1, Gpm6a 
       Lgals2, Smoc2, Serpinb6b, Slpi, Dpp4, Cldn15, Rspo1, Msln, Plxna4, Cybrd1 
       Aldh1a2, Nrgn, Fmod, Rpp25, Ogn, Hpgd, Prph, Adam33, Ptgis, Hacd4 
Negative:  Sparcl1, Pdgfra, Gsn, Mfap4, Htra3, Dpt, Mfap2, Col15a1, Itih5, Gpm6b 
       Ramp3, Lama2, Ntrk2, Penk, G0s2, Serpinf1, Spon1, Col6a3, Rxfp1, Plpp3 
       Lpl, Enpp2, Lhfp, Matn2, Ramp1, Nrp1, Atp1a2, Srpx, Emilin1, Pdgfrl 
PC_ 5 
Positive:  Pdgfra, Dcn, Dpt, Col15a1, Ramp3, Ntrk2, Lpl, Htra3, Htra1, Plxdc2 
       Aebp1, Cygb, Col5a3, Meg3, Lum, P2ry1, Fbln1, Dkk2, Pcolce, Dio2 
       Lrp1, Mfap5, Ctsk, Spon1, Col5a2, Col3a1, Ctsl, Matn2, Itih5, Rnase4 
Negative:  Myh11, Lmod1, Cnn1, Fbxl22, Acta2, Mustn1, Tagln, Actg2, Mcam, Sh3bgr 
       Des, Nexn, Myl9, Asb2, Ppp1r14a, Mylk, Tpm2, Synpo2, Myom1, Carmn 
       Slc22a3, Aoc3, Pdlim3, Slc2a4, Postn, Npnt, Tpm1, Dmpk, Smtn, Rbpms2 
proc.time()[3] - previous_time
elapsed 
  4.742 
DimPlot(sub_Estrus, reduction = "pca") + ggtitle(label = 'Immune cells removed')

ggsave(filename = "PCA_Immune_cells_removed.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image
FeaturePlot(sub_Estrus, reduction = "pca", features = c('Esr1', 'Ovgp1', 'Foxj1', 'Fxyd4', 'Ephx2', 'Dcn'))

ggsave(filename = "PCA_Immune_cells_removed1.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image
FeaturePlot(sub_Estrus, reduction = "pca", features = c('Fos', 'Kcne3', 'Serpina1e', 'Crabp2', 'Pgr', 'Vim'))

ggsave(filename = "PCA_Immune_cells_removed2.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image
previous_time <- proc.time()[3]
sub_Estrus_new <- FindNeighbors(sub_Estrus, dims = 1:22)
Computing nearest neighbor graph
Computing SNN
proc.time()[3] - previous_time
elapsed 
  0.375 
previous_time <- proc.time()[3]
sub_Estrus_new <- FindClusters(sub_Estrus_new, resolution = 0.04, algorithm = 4)
proc.time()[3] - previous_time
elapsed 
  0.711 
previous_time <- proc.time()[3]
sub_Estrus_new <- RunUMAP(sub_Estrus_new, dim = 1:22)
16:42:15 UMAP embedding parameters a = 0.9922 b = 1.112
16:42:15 Read 2318 rows and found 22 numeric columns
16:42:15 Using Annoy for neighbor search, n_neighbors = 30
16:42:15 Building Annoy index with metric = cosine, n_trees = 50
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
16:42:15 Writing NN index file to temp file /tmp/RtmprVQ6Uz/file1984921d941dd
16:42:15 Searching Annoy index using 1 thread, search_k = 3000
16:42:16 Annoy recall = 100%
16:42:16 Commencing smooth kNN distance calibration using 1 thread
16:42:16 Initializing from normalized Laplacian + noise
16:42:16 Commencing optimization for 500 epochs, with 91758 positive edges
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
16:42:18 Optimization finished
proc.time()[3] - previous_time
elapsed 
  3.395 
DimPlot(sub_Estrus_new, reduction = "umap") + ggtitle(label = 'Rescale UMAP after removing Immune cells')

ggsave(filename = "UMAP_Rescale.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 10 x 6.18 in image
FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Ovgp1', 'Foxj1', 'Crabp2', 'Myh11', 'Wt1', 'S100g', 'Sprr2f',
                  'Fxyd4'))

ggsave(filename = "UMAP_Rescale1.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image
FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Dcn', 'Pax8', 'Pax2', 'Twist2', 'Igf1', 'Serpina1e', 'Pdgfra', 'Krt19', 'Mki67'))

ggsave(filename = "UMAP_Rescale2.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image

Marker genes

marker_genes = c('Epcam', 'Krt15','Krt8','Krt19','Vim','Pdgfra','Twist2', 'Sprr2f', 'Fxyd4', 'Igfbp6',
                 'Acta2', 'Myh11','Foxj1','Ovgp1','Wt1','Serpina1e','S100g','Pecam1')

UMAP for marker genes

FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Epcam', 'Krt15','Krt8','Krt19','Vim','Pdgfra','Twist2', 'Sprr2f', 'Fxyd4'))

ggsave(filename = "UMAP_for_Marker_genes1.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image
FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Igfbp6',
                                                             'Acta2', 'Myh11','Foxj1','Ovgp1','Wt1','Serpina1e','S100g','Pecam1'))

ggsave(filename = "UMAP_for_Marker_genes2.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image

Rename clusters

Cluster.names <- c('1 Ciliated_InfAmp',
                   '2 Secretory_InfAmp',
                   '3 Epithelial_subset',
                   '4 Fibroblast Pdgfra+',
                   '5 Secretory_IsthUTJ',
                   '6 Fibroblast Pdgfra-',
                   '7 Muscle')
names(Cluster.names) <- levels(sub_Estrus_new)
sub_Estrus_new <- RenameIdents(sub_Estrus_new, Cluster.names)
DimPlot(sub_Estrus_new, reduction = "umap", pt.size = 0.5) + scale_color_manual(labels = Cluster.names,values=c('green',                                                                                        'red','blue','orange','grey','yellow','pink','purple'))
ggsave(filename = "UMAP_Clusters_names.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 7.29 x 4.5 in image

DotPlot(sub_Estrus_new, features = marker_genes, group.by = sub_Estrus_new@meta.data$ident) +  
  theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1)) +
  scale_y_discrete(labels = Cluster.names)

ggsave(filename = "DotPlot_for_Marker_genes.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 12 x 7.41 in image

Number of cells in each clusters

table(sub_Estrus_new@active.ident, sub_Estrus_new@meta.data$orig.ident)
                      
                       Estrus
  1 Ciliated_InfAmp       538
  2 Secretory_InfAmp      499
  3 Epithelial_subset     351
  4 Fibroblast Pdgfra+    347
  5 Secretory_IsthUTJ     304
  6 Fibroblast Pdgfra-    152
  7 Muscle                127

Extract top genes for each cluster

  • Default method of finding highly expressed genes for FindAllMarkers is Wilcoxon Rank Sum test
previous_time <- proc.time()[3]
sub_Estrus_new_markers <- FindAllMarkers(sub_Estrus_new, only.pos = TRUE, min.pct = 0.25, logfc.threshold = 0.25)
Calculating cluster 1 Ciliated_InfAmp

  |                                                  | 0 % ~calculating  
  |+                                                 | 1 % ~13s          
  |++                                                | 2 % ~13s          
  |++                                                | 3 % ~13s          
  |+++                                               | 4 % ~13s          
  |+++                                               | 5 % ~12s          
  |++++                                              | 6 % ~12s          
  |++++                                              | 7 % ~12s          
  |+++++                                             | 8 % ~12s          
  |+++++                                             | 9 % ~12s          
  |++++++                                            | 10% ~12s          
  |++++++                                            | 11% ~12s          
  |+++++++                                           | 12% ~11s          
  |+++++++                                           | 13% ~11s          
  |++++++++                                          | 14% ~11s          
  |++++++++                                          | 15% ~11s          
  |+++++++++                                         | 16% ~11s          
  |+++++++++                                         | 18% ~11s          
  |++++++++++                                        | 19% ~11s          
  |++++++++++                                        | 20% ~11s          
  |+++++++++++                                       | 21% ~10s          
  |+++++++++++                                       | 22% ~10s          
  |++++++++++++                                      | 23% ~10s          
  |++++++++++++                                      | 24% ~10s          
  |+++++++++++++                                     | 25% ~10s          
  |+++++++++++++                                     | 26% ~10s          
  |++++++++++++++                                    | 27% ~10s          
  |++++++++++++++                                    | 28% ~10s          
  |+++++++++++++++                                   | 29% ~09s          
  |+++++++++++++++                                   | 30% ~09s          
  |++++++++++++++++                                  | 31% ~09s          
  |++++++++++++++++                                  | 32% ~09s          
  |+++++++++++++++++                                 | 33% ~09s          
  |++++++++++++++++++                                | 34% ~09s          
  |++++++++++++++++++                                | 35% ~09s          
  |+++++++++++++++++++                               | 36% ~08s          
  |+++++++++++++++++++                               | 37% ~08s          
  |++++++++++++++++++++                              | 38% ~08s          
  |++++++++++++++++++++                              | 39% ~08s          
  |+++++++++++++++++++++                             | 40% ~08s          
  |+++++++++++++++++++++                             | 41% ~08s          
  |++++++++++++++++++++++                            | 42% ~08s          
  |++++++++++++++++++++++                            | 43% ~07s          
  |+++++++++++++++++++++++                           | 44% ~07s          
  |+++++++++++++++++++++++                           | 45% ~07s          
  |++++++++++++++++++++++++                          | 46% ~07s          
  |++++++++++++++++++++++++                          | 47% ~07s          
  |+++++++++++++++++++++++++                         | 48% ~07s          
  |+++++++++++++++++++++++++                         | 49% ~07s          
  |++++++++++++++++++++++++++                        | 51% ~07s          
  |++++++++++++++++++++++++++                        | 52% ~06s          
  |+++++++++++++++++++++++++++                       | 53% ~06s          
  |+++++++++++++++++++++++++++                       | 54% ~06s          
  |++++++++++++++++++++++++++++                      | 55% ~06s          
  |++++++++++++++++++++++++++++                      | 56% ~06s          
  |+++++++++++++++++++++++++++++                     | 57% ~06s          
  |+++++++++++++++++++++++++++++                     | 58% ~06s          
  |++++++++++++++++++++++++++++++                    | 59% ~05s          
  |++++++++++++++++++++++++++++++                    | 60% ~05s          
  |+++++++++++++++++++++++++++++++                   | 61% ~05s          
  |+++++++++++++++++++++++++++++++                   | 62% ~05s          
  |++++++++++++++++++++++++++++++++                  | 63% ~05s          
  |++++++++++++++++++++++++++++++++                  | 64% ~05s          
  |+++++++++++++++++++++++++++++++++                 | 65% ~05s          
  |+++++++++++++++++++++++++++++++++                 | 66% ~04s          
  |++++++++++++++++++++++++++++++++++                | 67% ~04s          
  |+++++++++++++++++++++++++++++++++++               | 68% ~04s          
  |+++++++++++++++++++++++++++++++++++               | 69% ~04s          
  |++++++++++++++++++++++++++++++++++++              | 70% ~04s          
  |++++++++++++++++++++++++++++++++++++              | 71% ~04s          
  |+++++++++++++++++++++++++++++++++++++             | 72% ~04s          
  |+++++++++++++++++++++++++++++++++++++             | 73% ~04s          
  |++++++++++++++++++++++++++++++++++++++            | 74% ~03s          
  |++++++++++++++++++++++++++++++++++++++            | 75% ~03s          
  |+++++++++++++++++++++++++++++++++++++++           | 76% ~03s          
  |+++++++++++++++++++++++++++++++++++++++           | 77% ~03s          
  |++++++++++++++++++++++++++++++++++++++++          | 78% ~03s          
  |++++++++++++++++++++++++++++++++++++++++          | 79% ~03s          
  |+++++++++++++++++++++++++++++++++++++++++         | 80% ~03s          
  |+++++++++++++++++++++++++++++++++++++++++         | 81% ~02s          
  |++++++++++++++++++++++++++++++++++++++++++        | 82% ~02s          
  |++++++++++++++++++++++++++++++++++++++++++        | 84% ~02s          
  |+++++++++++++++++++++++++++++++++++++++++++       | 85% ~02s          
  |+++++++++++++++++++++++++++++++++++++++++++       | 86% ~02s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 87% ~02s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 88% ~02s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 89% ~02s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 92% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 93% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 94% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 95% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 96% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 97% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=13s  
Calculating cluster 2 Secretory_InfAmp

  |                                                  | 0 % ~calculating  
  |+                                                 | 1 % ~04s          
  |++                                                | 2 % ~04s          
  |++                                                | 3 % ~03s          
  |+++                                               | 4 % ~03s          
  |+++                                               | 5 % ~03s          
  |++++                                              | 6 % ~03s          
  |++++                                              | 8 % ~03s          
  |+++++                                             | 9 % ~03s          
  |+++++                                             | 10% ~03s          
  |++++++                                            | 11% ~03s          
  |++++++                                            | 12% ~03s          
  |+++++++                                           | 13% ~03s          
  |+++++++                                           | 14% ~03s          
  |++++++++                                          | 15% ~03s          
  |+++++++++                                         | 16% ~03s          
  |+++++++++                                         | 17% ~03s          
  |++++++++++                                        | 18% ~03s          
  |++++++++++                                        | 19% ~03s          
  |+++++++++++                                       | 20% ~03s          
  |+++++++++++                                       | 22% ~03s          
  |++++++++++++                                      | 23% ~03s          
  |++++++++++++                                      | 24% ~03s          
  |+++++++++++++                                     | 25% ~03s          
  |+++++++++++++                                     | 26% ~03s          
  |++++++++++++++                                    | 27% ~03s          
  |++++++++++++++                                    | 28% ~03s          
  |+++++++++++++++                                   | 29% ~03s          
  |++++++++++++++++                                  | 30% ~03s          
  |++++++++++++++++                                  | 31% ~03s          
  |+++++++++++++++++                                 | 32% ~02s          
  |+++++++++++++++++                                 | 33% ~02s          
  |++++++++++++++++++                                | 34% ~02s          
  |++++++++++++++++++                                | 35% ~02s          
  |+++++++++++++++++++                               | 37% ~02s          
  |+++++++++++++++++++                               | 38% ~02s          
  |++++++++++++++++++++                              | 39% ~02s          
  |++++++++++++++++++++                              | 40% ~02s          
  |+++++++++++++++++++++                             | 41% ~02s          
  |+++++++++++++++++++++                             | 42% ~02s          
  |++++++++++++++++++++++                            | 43% ~02s          
  |+++++++++++++++++++++++                           | 44% ~02s          
  |+++++++++++++++++++++++                           | 45% ~02s          
  |++++++++++++++++++++++++                          | 46% ~02s          
  |++++++++++++++++++++++++                          | 47% ~02s          
  |+++++++++++++++++++++++++                         | 48% ~02s          
  |+++++++++++++++++++++++++                         | 49% ~02s          
  |++++++++++++++++++++++++++                        | 51% ~02s          
  |++++++++++++++++++++++++++                        | 52% ~02s          
  |+++++++++++++++++++++++++++                       | 53% ~02s          
  |+++++++++++++++++++++++++++                       | 54% ~02s          
  |++++++++++++++++++++++++++++                      | 55% ~02s          
  |++++++++++++++++++++++++++++                      | 56% ~02s          
  |+++++++++++++++++++++++++++++                     | 57% ~02s          
  |++++++++++++++++++++++++++++++                    | 58% ~02s          
  |++++++++++++++++++++++++++++++                    | 59% ~01s          
  |+++++++++++++++++++++++++++++++                   | 60% ~01s          
  |+++++++++++++++++++++++++++++++                   | 61% ~01s          
  |++++++++++++++++++++++++++++++++                  | 62% ~01s          
  |++++++++++++++++++++++++++++++++                  | 63% ~01s          
  |+++++++++++++++++++++++++++++++++                 | 65% ~01s          
  |+++++++++++++++++++++++++++++++++                 | 66% ~01s          
  |++++++++++++++++++++++++++++++++++                | 67% ~01s          
  |++++++++++++++++++++++++++++++++++                | 68% ~01s          
  |+++++++++++++++++++++++++++++++++++               | 69% ~01s          
  |+++++++++++++++++++++++++++++++++++               | 70% ~01s          
  |++++++++++++++++++++++++++++++++++++              | 71% ~01s          
  |+++++++++++++++++++++++++++++++++++++             | 72% ~01s          
  |+++++++++++++++++++++++++++++++++++++             | 73% ~01s          
  |++++++++++++++++++++++++++++++++++++++            | 74% ~01s          
  |++++++++++++++++++++++++++++++++++++++            | 75% ~01s          
  |+++++++++++++++++++++++++++++++++++++++           | 76% ~01s          
  |+++++++++++++++++++++++++++++++++++++++           | 77% ~01s          
  |++++++++++++++++++++++++++++++++++++++++          | 78% ~01s          
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++         | 81% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++         | 82% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++        | 83% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++        | 84% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++       | 85% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 86% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 87% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 88% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 89% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 90% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 92% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 94% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 95% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 96% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 97% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=04s  
Calculating cluster 3 Epithelial_subset

  |                                                  | 0 % ~calculating  
  |+                                                 | 1 % ~06s          
  |++                                                | 2 % ~04s          
  |++                                                | 3 % ~04s          
  |+++                                               | 4 % ~04s          
  |+++                                               | 5 % ~03s          
  |++++                                              | 6 % ~03s          
  |++++                                              | 8 % ~03s          
  |+++++                                             | 9 % ~03s          
  |+++++                                             | 10% ~03s          
  |++++++                                            | 11% ~03s          
  |++++++                                            | 12% ~03s          
  |+++++++                                           | 13% ~03s          
  |+++++++                                           | 14% ~03s          
  |++++++++                                          | 15% ~03s          
  |+++++++++                                         | 16% ~03s          
  |+++++++++                                         | 17% ~03s          
  |++++++++++                                        | 18% ~03s          
  |++++++++++                                        | 19% ~03s          
  |+++++++++++                                       | 20% ~03s          
  |+++++++++++                                       | 22% ~03s          
  |++++++++++++                                      | 23% ~03s          
  |++++++++++++                                      | 24% ~03s          
  |+++++++++++++                                     | 25% ~02s          
  |+++++++++++++                                     | 26% ~02s          
  |++++++++++++++                                    | 27% ~02s          
  |++++++++++++++                                    | 28% ~02s          
  |+++++++++++++++                                   | 29% ~02s          
  |++++++++++++++++                                  | 30% ~02s          
  |++++++++++++++++                                  | 31% ~02s          
  |+++++++++++++++++                                 | 32% ~02s          
  |+++++++++++++++++                                 | 33% ~02s          
  |++++++++++++++++++                                | 34% ~02s          
  |++++++++++++++++++                                | 35% ~02s          
  |+++++++++++++++++++                               | 37% ~02s          
  |+++++++++++++++++++                               | 38% ~02s          
  |++++++++++++++++++++                              | 39% ~02s          
  |++++++++++++++++++++                              | 40% ~02s          
  |+++++++++++++++++++++                             | 41% ~02s          
  |+++++++++++++++++++++                             | 42% ~02s          
  |++++++++++++++++++++++                            | 43% ~02s          
  |+++++++++++++++++++++++                           | 44% ~02s          
  |+++++++++++++++++++++++                           | 45% ~02s          
  |++++++++++++++++++++++++                          | 46% ~02s          
  |++++++++++++++++++++++++                          | 47% ~02s          
  |+++++++++++++++++++++++++                         | 48% ~02s          
  |+++++++++++++++++++++++++                         | 49% ~02s          
  |++++++++++++++++++++++++++                        | 51% ~02s          
  |++++++++++++++++++++++++++                        | 52% ~02s          
  |+++++++++++++++++++++++++++                       | 53% ~02s          
  |+++++++++++++++++++++++++++                       | 54% ~02s          
  |++++++++++++++++++++++++++++                      | 55% ~01s          
  |++++++++++++++++++++++++++++                      | 56% ~01s          
  |+++++++++++++++++++++++++++++                     | 57% ~01s          
  |++++++++++++++++++++++++++++++                    | 58% ~01s          
  |++++++++++++++++++++++++++++++                    | 59% ~01s          
  |+++++++++++++++++++++++++++++++                   | 60% ~01s          
  |+++++++++++++++++++++++++++++++                   | 61% ~01s          
  |++++++++++++++++++++++++++++++++                  | 62% ~01s          
  |++++++++++++++++++++++++++++++++                  | 63% ~01s          
  |+++++++++++++++++++++++++++++++++                 | 65% ~01s          
  |+++++++++++++++++++++++++++++++++                 | 66% ~01s          
  |++++++++++++++++++++++++++++++++++                | 67% ~01s          
  |++++++++++++++++++++++++++++++++++                | 68% ~01s          
  |+++++++++++++++++++++++++++++++++++               | 69% ~01s          
  |+++++++++++++++++++++++++++++++++++               | 70% ~01s          
  |++++++++++++++++++++++++++++++++++++              | 71% ~01s          
  |+++++++++++++++++++++++++++++++++++++             | 72% ~01s          
  |+++++++++++++++++++++++++++++++++++++             | 73% ~01s          
  |++++++++++++++++++++++++++++++++++++++            | 74% ~01s          
  |++++++++++++++++++++++++++++++++++++++            | 75% ~01s          
  |+++++++++++++++++++++++++++++++++++++++           | 76% ~01s          
  |+++++++++++++++++++++++++++++++++++++++           | 77% ~01s          
  |++++++++++++++++++++++++++++++++++++++++          | 78% ~01s          
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++         | 81% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++         | 82% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++        | 83% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++        | 84% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++       | 85% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 86% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 87% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 88% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 89% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 90% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 92% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 94% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 95% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 96% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 97% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03s  
Calculating cluster 4 Fibroblast Pdgfra+

  |                                                  | 0 % ~calculating  
  |+                                                 | 1 % ~03s          
  |++                                                | 2 % ~03s          
  |++                                                | 3 % ~03s          
  |+++                                               | 4 % ~03s          
  |+++                                               | 5 % ~03s          
  |++++                                              | 6 % ~03s          
  |++++                                              | 8 % ~03s          
  |+++++                                             | 9 % ~03s          
  |+++++                                             | 10% ~03s          
  |++++++                                            | 11% ~03s          
  |++++++                                            | 12% ~03s          
  |+++++++                                           | 13% ~03s          
  |+++++++                                           | 14% ~03s          
  |++++++++                                          | 15% ~03s          
  |+++++++++                                         | 16% ~03s          
  |+++++++++                                         | 17% ~03s          
  |++++++++++                                        | 18% ~02s          
  |++++++++++                                        | 19% ~02s          
  |+++++++++++                                       | 20% ~02s          
  |+++++++++++                                       | 22% ~02s          
  |++++++++++++                                      | 23% ~02s          
  |++++++++++++                                      | 24% ~02s          
  |+++++++++++++                                     | 25% ~02s          
  |+++++++++++++                                     | 26% ~02s          
  |++++++++++++++                                    | 27% ~02s          
  |++++++++++++++                                    | 28% ~02s          
  |+++++++++++++++                                   | 29% ~02s          
  |++++++++++++++++                                  | 30% ~02s          
  |++++++++++++++++                                  | 31% ~02s          
  |+++++++++++++++++                                 | 32% ~02s          
  |+++++++++++++++++                                 | 33% ~02s          
  |++++++++++++++++++                                | 34% ~02s          
  |++++++++++++++++++                                | 35% ~02s          
  |+++++++++++++++++++                               | 37% ~02s          
  |+++++++++++++++++++                               | 38% ~02s          
  |++++++++++++++++++++                              | 39% ~02s          
  |++++++++++++++++++++                              | 40% ~02s          
  |+++++++++++++++++++++                             | 41% ~02s          
  |+++++++++++++++++++++                             | 42% ~02s          
  |++++++++++++++++++++++                            | 43% ~02s          
  |+++++++++++++++++++++++                           | 44% ~02s          
  |+++++++++++++++++++++++                           | 45% ~02s          
  |++++++++++++++++++++++++                          | 46% ~02s          
  |++++++++++++++++++++++++                          | 47% ~02s          
  |+++++++++++++++++++++++++                         | 48% ~02s          
  |+++++++++++++++++++++++++                         | 49% ~02s          
  |++++++++++++++++++++++++++                        | 51% ~01s          
  |++++++++++++++++++++++++++                        | 52% ~01s          
  |+++++++++++++++++++++++++++                       | 53% ~01s          
  |+++++++++++++++++++++++++++                       | 54% ~01s          
  |++++++++++++++++++++++++++++                      | 55% ~01s          
  |++++++++++++++++++++++++++++                      | 56% ~01s          
  |+++++++++++++++++++++++++++++                     | 57% ~01s          
  |++++++++++++++++++++++++++++++                    | 58% ~01s          
  |++++++++++++++++++++++++++++++                    | 59% ~01s          
  |+++++++++++++++++++++++++++++++                   | 60% ~01s          
  |+++++++++++++++++++++++++++++++                   | 61% ~01s          
  |++++++++++++++++++++++++++++++++                  | 62% ~01s          
  |++++++++++++++++++++++++++++++++                  | 63% ~01s          
  |+++++++++++++++++++++++++++++++++                 | 65% ~01s          
  |+++++++++++++++++++++++++++++++++                 | 66% ~01s          
  |++++++++++++++++++++++++++++++++++                | 67% ~01s          
  |++++++++++++++++++++++++++++++++++                | 68% ~01s          
  |+++++++++++++++++++++++++++++++++++               | 69% ~01s          
  |+++++++++++++++++++++++++++++++++++               | 70% ~01s          
  |++++++++++++++++++++++++++++++++++++              | 71% ~01s          
  |+++++++++++++++++++++++++++++++++++++             | 72% ~01s          
  |+++++++++++++++++++++++++++++++++++++             | 73% ~01s          
  |++++++++++++++++++++++++++++++++++++++            | 74% ~01s          
  |++++++++++++++++++++++++++++++++++++++            | 75% ~01s          
  |+++++++++++++++++++++++++++++++++++++++           | 76% ~01s          
  |+++++++++++++++++++++++++++++++++++++++           | 77% ~01s          
  |++++++++++++++++++++++++++++++++++++++++          | 78% ~01s          
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++         | 81% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++         | 82% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++        | 83% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++        | 84% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++       | 85% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 86% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 87% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 88% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 89% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 90% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 92% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 94% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 95% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 96% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 97% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03s  
Calculating cluster 5 Secretory_IsthUTJ

  |                                                  | 0 % ~calculating  
  |+                                                 | 1 % ~05s          
  |++                                                | 2 % ~05s          
  |++                                                | 3 % ~04s          
  |+++                                               | 4 % ~04s          
  |+++                                               | 5 % ~04s          
  |++++                                              | 6 % ~04s          
  |++++                                              | 7 % ~04s          
  |+++++                                             | 8 % ~04s          
  |+++++                                             | 9 % ~04s          
  |++++++                                            | 10% ~04s          
  |++++++                                            | 11% ~04s          
  |+++++++                                           | 12% ~04s          
  |+++++++                                           | 14% ~04s          
  |++++++++                                          | 15% ~04s          
  |++++++++                                          | 16% ~04s          
  |+++++++++                                         | 17% ~04s          
  |+++++++++                                         | 18% ~04s          
  |++++++++++                                        | 19% ~04s          
  |++++++++++                                        | 20% ~04s          
  |+++++++++++                                       | 21% ~04s          
  |+++++++++++                                       | 22% ~04s          
  |++++++++++++                                      | 23% ~03s          
  |++++++++++++                                      | 24% ~03s          
  |+++++++++++++                                     | 25% ~03s          
  |++++++++++++++                                    | 26% ~03s          
  |++++++++++++++                                    | 27% ~03s          
  |+++++++++++++++                                   | 28% ~03s          
  |+++++++++++++++                                   | 29% ~03s          
  |++++++++++++++++                                  | 30% ~03s          
  |++++++++++++++++                                  | 31% ~03s          
  |+++++++++++++++++                                 | 32% ~03s          
  |+++++++++++++++++                                 | 33% ~03s          
  |++++++++++++++++++                                | 34% ~03s          
  |++++++++++++++++++                                | 35% ~03s          
  |+++++++++++++++++++                               | 36% ~03s          
  |+++++++++++++++++++                               | 38% ~03s          
  |++++++++++++++++++++                              | 39% ~03s          
  |++++++++++++++++++++                              | 40% ~03s          
  |+++++++++++++++++++++                             | 41% ~03s          
  |+++++++++++++++++++++                             | 42% ~03s          
  |++++++++++++++++++++++                            | 43% ~03s          
  |++++++++++++++++++++++                            | 44% ~03s          
  |+++++++++++++++++++++++                           | 45% ~03s          
  |+++++++++++++++++++++++                           | 46% ~02s          
  |++++++++++++++++++++++++                          | 47% ~02s          
  |++++++++++++++++++++++++                          | 48% ~02s          
  |+++++++++++++++++++++++++                         | 49% ~02s          
  |+++++++++++++++++++++++++                         | 50% ~02s          
  |++++++++++++++++++++++++++                        | 51% ~02s          
  |+++++++++++++++++++++++++++                       | 52% ~02s          
  |+++++++++++++++++++++++++++                       | 53% ~02s          
  |++++++++++++++++++++++++++++                      | 54% ~02s          
  |++++++++++++++++++++++++++++                      | 55% ~02s          
  |+++++++++++++++++++++++++++++                     | 56% ~02s          
  |+++++++++++++++++++++++++++++                     | 57% ~02s          
  |++++++++++++++++++++++++++++++                    | 58% ~02s          
  |++++++++++++++++++++++++++++++                    | 59% ~02s          
  |+++++++++++++++++++++++++++++++                   | 60% ~02s          
  |+++++++++++++++++++++++++++++++                   | 61% ~02s          
  |++++++++++++++++++++++++++++++++                  | 62% ~02s          
  |++++++++++++++++++++++++++++++++                  | 64% ~02s          
  |+++++++++++++++++++++++++++++++++                 | 65% ~02s          
  |+++++++++++++++++++++++++++++++++                 | 66% ~02s          
  |++++++++++++++++++++++++++++++++++                | 67% ~02s          
  |++++++++++++++++++++++++++++++++++                | 68% ~01s          
  |+++++++++++++++++++++++++++++++++++               | 69% ~01s          
  |+++++++++++++++++++++++++++++++++++               | 70% ~01s          
  |++++++++++++++++++++++++++++++++++++              | 71% ~01s          
  |++++++++++++++++++++++++++++++++++++              | 72% ~01s          
  |+++++++++++++++++++++++++++++++++++++             | 73% ~01s          
  |+++++++++++++++++++++++++++++++++++++             | 74% ~01s          
  |++++++++++++++++++++++++++++++++++++++            | 75% ~01s          
  |+++++++++++++++++++++++++++++++++++++++           | 76% ~01s          
  |+++++++++++++++++++++++++++++++++++++++           | 77% ~01s          
  |++++++++++++++++++++++++++++++++++++++++          | 78% ~01s          
  |++++++++++++++++++++++++++++++++++++++++          | 79% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++         | 80% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++         | 81% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++        | 82% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++        | 83% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++       | 84% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++       | 85% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 86% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 88% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 89% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 92% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 93% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 94% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 95% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 96% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 97% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=05s  
Calculating cluster 6 Fibroblast Pdgfra-

  |                                                  | 0 % ~calculating  
  |+                                                 | 1 % ~04s          
  |++                                                | 2 % ~04s          
  |++                                                | 3 % ~04s          
  |+++                                               | 4 % ~04s          
  |+++                                               | 5 % ~04s          
  |++++                                              | 6 % ~04s          
  |++++                                              | 8 % ~04s          
  |+++++                                             | 9 % ~04s          
  |+++++                                             | 10% ~04s          
  |++++++                                            | 11% ~04s          
  |++++++                                            | 12% ~04s          
  |+++++++                                           | 13% ~04s          
  |+++++++                                           | 14% ~04s          
  |++++++++                                          | 15% ~04s          
  |+++++++++                                         | 16% ~04s          
  |+++++++++                                         | 17% ~04s          
  |++++++++++                                        | 18% ~04s          
  |++++++++++                                        | 19% ~04s          
  |+++++++++++                                       | 20% ~03s          
  |+++++++++++                                       | 22% ~03s          
  |++++++++++++                                      | 23% ~03s          
  |++++++++++++                                      | 24% ~03s          
  |+++++++++++++                                     | 25% ~03s          
  |+++++++++++++                                     | 26% ~03s          
  |++++++++++++++                                    | 27% ~03s          
  |++++++++++++++                                    | 28% ~03s          
  |+++++++++++++++                                   | 29% ~03s          
  |++++++++++++++++                                  | 30% ~03s          
  |++++++++++++++++                                  | 31% ~03s          
  |+++++++++++++++++                                 | 32% ~03s          
  |+++++++++++++++++                                 | 33% ~03s          
  |++++++++++++++++++                                | 34% ~03s          
  |++++++++++++++++++                                | 35% ~03s          
  |+++++++++++++++++++                               | 37% ~03s          
  |+++++++++++++++++++                               | 38% ~03s          
  |++++++++++++++++++++                              | 39% ~03s          
  |++++++++++++++++++++                              | 40% ~03s          
  |+++++++++++++++++++++                             | 41% ~03s          
  |+++++++++++++++++++++                             | 42% ~03s          
  |++++++++++++++++++++++                            | 43% ~02s          
  |+++++++++++++++++++++++                           | 44% ~02s          
  |+++++++++++++++++++++++                           | 45% ~02s          
  |++++++++++++++++++++++++                          | 46% ~02s          
  |++++++++++++++++++++++++                          | 47% ~02s          
  |+++++++++++++++++++++++++                         | 48% ~02s          
  |+++++++++++++++++++++++++                         | 49% ~02s          
  |++++++++++++++++++++++++++                        | 51% ~02s          
  |++++++++++++++++++++++++++                        | 52% ~02s          
  |+++++++++++++++++++++++++++                       | 53% ~02s          
  |+++++++++++++++++++++++++++                       | 54% ~02s          
  |++++++++++++++++++++++++++++                      | 55% ~02s          
  |++++++++++++++++++++++++++++                      | 56% ~02s          
  |+++++++++++++++++++++++++++++                     | 57% ~02s          
  |++++++++++++++++++++++++++++++                    | 58% ~02s          
  |++++++++++++++++++++++++++++++                    | 59% ~02s          
  |+++++++++++++++++++++++++++++++                   | 60% ~02s          
  |+++++++++++++++++++++++++++++++                   | 61% ~02s          
  |++++++++++++++++++++++++++++++++                  | 62% ~02s          
  |++++++++++++++++++++++++++++++++                  | 63% ~02s          
  |+++++++++++++++++++++++++++++++++                 | 65% ~02s          
  |+++++++++++++++++++++++++++++++++                 | 66% ~01s          
  |++++++++++++++++++++++++++++++++++                | 67% ~01s          
  |++++++++++++++++++++++++++++++++++                | 68% ~01s          
  |+++++++++++++++++++++++++++++++++++               | 69% ~01s          
  |+++++++++++++++++++++++++++++++++++               | 70% ~01s          
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sub_Estrus_new_markers %>%
    group_by(cluster) %>%
    slice_max(n = 20, order_by = avg_log2FC)
proc.time()[3] - previous_time
elapsed 
  38.09 

Heat map for top 10 genes in each cluster

sub_Estrus_new_markers %>%
    group_by(cluster) %>%
    top_n(n = 10, wt = avg_log2FC) -> top10
DoHeatmap(sub_Estrus_new, features = top10$gene) + NoLegend()

ggsave(filename = "HeatMap_Top10_genes.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
Saving 10 x 6.18 in image
saveRDS(sub_Estrus_new, file = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Estrus_subset_analyzed.rds")
---
title: "scRNA-seq analysis using Seurat package"
output: html_notebook
author: Long Nguyen
---
# Description:
* The purpose of this notebook is to analyze scRNA-seq data of mouse Estrus
* The method used is reading directly from mtx files (CellRanger count outputs)
* The algorithms used come from Seurat package
* Two mitochondiral genes: mt-Nd6 and mt-Cytb will be manually deleted
* Because read_loom method in Scanpy does not contain those genes after filtering was done
* The results will compared to:
  + Same dataset analyzed using Scanpy - ManuallyRemoveGenes
  + Same dataset analyzed using Scanpy - readloom
  
# Workflow begin
This tutorial comes from [Seurat](https://satijalab.org/seurat/articles/pbmc3k_tutorial.html)

## Load required packages
```{r}
library(dplyr)
library(Seurat)
library(leiden)
library(ggplot2)
library(patchwork)
```


## Load data
Input is a directory that contains CellRanger count outputs
```{r}
previous_time <- proc.time()[3]
Estrus.data <-Read10X(data.dir ="/home/nguyen/Estrus_cellranger_count/Estrus_Raw/")
proc.time()[3] - previous_time
```
* View(Estrus.data) - run this command to investigate the output
* Current dataset:
  + 31253 genes
  + 6599 cells

## Initialize the Seurat object
* Initialize the Seurat object with the raw (non-normalized data).
* Filter out genes that are detected in less than 3 cells 
* Filter out cells that have less than 200 genes expressed
```{r}
previous_time <- proc.time()[3]
Estrus <- CreateSeuratObject(counts = Estrus.data, project = "Estrus", min.cells = 3, min.features = 200)
proc.time()[3] - previous_time
Estrus
```
* Output:
  + 19420 genes 
  + 6481 cells

## Mitochondiral genes overview
```{r}
# This is the list of genes that start with mt
mito_genes <- grep("^mt-", rownames(Estrus) , ignore.case=T, value=T)
print(mito_genes)
```

## mt-Nd6 and mt-Cytb will be manually deleted
```{r}
counts <- GetAssayData(Estrus, assay = "RNA")
counts <- counts[-(which(rownames(counts) %in% c("mt-Nd6", "mt-Cytb"))),]
Estrus <- subset(Estrus, features = rownames(counts))
```

```{r}
# This is the list of genes that start with mt
mito_genes <- grep("^mt-", rownames(Estrus) , ignore.case=T, value=T)
print(mito_genes)
```
* "mt-Nd6", "mt-Cytb" were deleted

## Calculate the percentage of mitochondrial genes
* Add "percent.mt" column to object metadata
* Percentage of mitochondrial genes is stored in "percent.mt"
* "^mt-" means strings start with mt (lowercase mt for mice, uppercase MT for human)
```{r}
Estrus[["percent.mt"]] <- PercentageFeatureSet(Estrus, pattern = "^mt-")
```

```{r}
# This is the data in "percent.mt" column
percent_mito <- FetchData(object = Estrus, vars = "percent.mt") #how to fetch data
head(percent_mito)
```

```{r, fig.width=10}
# Visualize QC metrics as a violin plot
VlnPlot(Estrus, features = c("nFeature_RNA", "nCount_RNA", "percent.mt"), ncol = 3)
```
```{r}
# FeatureScatter is typically used to visualize feature-feature relationships, but can be used
# for anything calculated by the object, i.e. columns in object metadata, PC scores etc.
plot1 <- FeatureScatter(Estrus, feature1 = "nCount_RNA", feature2 = "percent.mt")
plot2 <- FeatureScatter(Estrus, feature1 = "nCount_RNA", feature2 = "nFeature_RNA")
plot1 + plot2
```

## Filter out low-quality cells
* We filter out cells that have unique feature counts over 6000
* Because any cell that has more than 6000 genes are likely doublets
* Note: Seurat tutorial used 2500 instead of 6000
* We also filter out cells that have more than 5% mitochondrial genes
* Because those are likely to be nonviable cells
```{r}
previous_time <- proc.time()[3]
Estrus <- subset(Estrus, subset = nFeature_RNA < 6000 & percent.mt < 5)
proc.time()[3] - previous_time
```
```{r}
Estrus
```
* After filtering step, the dataset is left with
 + 19418 genes
 + 2585 cells

## Normalizing the data
* Perform log-normalization method
* Feature counts for each cell are divided by the total counts for that cell and multiplied by the scale.factor
* scale.factor default value = 10,000
* This is then natural-log transformed using log1p
```{r}
previous_time <- proc.time()[3]
Estrus <- NormalizeData(Estrus, normalization.method = "LogNormalize", scale.factor = 10000)
proc.time()[3] - previous_time
```
## Identification of highly variable features (feature selection)
```{r}
previous_time <- proc.time()[3]
Estrus <- FindVariableFeatures(Estrus, selection.method = "disp", nfeatures = 5000)
proc.time()[3] - previous_time
```
## Plotting highly variable features
```{r}
# Identify the 10 most highly variable genes
top10 <- head(VariableFeatures(Estrus), 10)
top10
```

```{r, fig.width=10, fig.height=4}
# Plot variable features with and without labels
plot1 <- VariableFeaturePlot(Estrus)
plot2 <- LabelPoints(plot = plot1, points = top10, repel = TRUE)
plot1 + plot2 
```
## Regress out data and perform scaling
* Regressing out total counts per cell and percent.mt before scaling. 
* Take out mitochondrial genes before scaling (analyzing) the data 
* Because mito_genes shouldn't be accounted for cell population analysis.
```{r}
previous_time <- proc.time()[3]
all.genes <- rownames(Estrus)
Estrus <- ScaleData(Estrus, features = all.genes, vars.to.regress = c("nCount_RNA","percent.mt"), scale.max = 10)
# Explanations below are from [Seurat](https://satijalab.org/seurat/archive/v3.0/cell_cycle_vignette.html)
# vars.to.regress = RegressOut in Seurat version 1.4 or lower
# However, as the results of this procedure are stored in the scaled data slot (therefore overwriting the output of ScaleData) 
# This functionality is merged into the ScaleData function itself.
proc.time()[3] - previous_time
```
## Perform linear dimensional reduction (PCA)
```{r}
previous_time <- proc.time()[3]
Estrus <- RunPCA(Estrus, features = VariableFeatures(object = Estrus))
proc.time()[3] - previous_time
```

## Examine and visualize PCA results a few different ways
```{r}
print(Estrus[["pca"]], dims = 1:5, nfeatures = 5)
```

```{r}
VizDimLoadings(Estrus, dims = 1:2, reduction = "pca")
```

```{r}
DimPlot(Estrus, reduction = "pca") + ggtitle(label = 'Including immune cells') 
ggsave(filename = "PCA_Including_immune_cells.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```

```{r, fig.width=12}
FeaturePlot(Estrus, reduction = "pca", features = c('Ovgp1', 
                                                    'Foxj1', 'Igf1', 'Pdxk', 
                                                    'Fxyd4', 'Bsg', 
                                                    'Serpina1e', 'Krt19'))
ggsave(filename = "PCA_Including_immune_cells1.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```
```{r, fig.width=12}
FeaturePlot(Estrus, reduction = "pca", features = c('Dcn', 'Pax8',
                                                    'Ephx2', 
                                                    'Pecam1', 'Pdgfra', 'Myh11', 'Atp2b4', 
                                                    'Cd52'))
ggsave(filename = "PCA_Including_immune_cells2.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```

## Determine the ‘dimensionality’ of the data
* Seurat tutorial uses JackStraw() and and ScoreJackStraw to determine dimensionality
* the syntax is: 
  + Estrus <- JackStraw(Estrus, num.replicate = 100)
  + Estrus <- ScoreJackStraw(Estrus, dims = 1:20)
* However, it takes very long. 
* An elbow plot often corresponds well enough with the significant dims and is much faster to run than Jackstraw
* According to the graph below, choose the first 22 principle components to use
```{r}
ElbowPlot(Estrus, ndims = 40)
```
## Find neighbors
* Dims = dimensions = determined as 22 from the above step
```{r}
previous_time <- proc.time()[3]
Estrus_new <- FindNeighbors(Estrus, dims = 1:22, k.param = 10)
proc.time()[3] - previous_time
```
## Find cell clusters using Leiden's method
```{r}
# Algorithm = 4 specifies Leiden's method
previous_time <- proc.time()[3]
Estrus_new <- FindClusters(Estrus_new, resolution = 0.14, algorithm = 4)
proc.time()[3] - previous_time
```
## Constructing UMAP plot
```{r}
previous_time <- proc.time()[3]
Estrus_new <- RunUMAP(Estrus_new, dim = 1:22) 
proc.time()[3] - previous_time
```
## Visualization
```{r}
DimPlot(Estrus_new, reduction = "umap") + ggtitle(label = 'Including immune cells')
ggsave(filename = "UMAP_Including_immune_cells.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```
```{r, fig.width=12}
FeaturePlot(Estrus_new, reduction = "umap", features = c('Ovgp1', 'Foxj1', 'Dcn', 'Serpina1e', 'Sprr2f',
                                                         'Fxyd4', 'Ly6a', 'Epcam', 'Krt19', 'Vim', 'Pdgfra'))
ggsave(filename = "UMAP_Including_immune_cells1.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```

```{r, fig.width=12}
FeaturePlot(Estrus_new, reduction = "umap", features = c('Myh11', 
                                        'Dkk2', 'Slitrk6', 'Dio2', 'Smoc2', 'Tes', 'Dab1', 'Camk1d', 'Pecam1', 'Cd52','Mki67'))
ggsave(filename = "UMAP_Including_immune_cells2.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```
## Removing immune cells
```{r, fig.width=12}
sub_Estrus = subset(x = Estrus_new, ident = c(1,2,3,4,5,6,7,9,10,12))
DimPlot(sub_Estrus, reduction = "umap") + ggtitle(label = 'Immune cells removed')
ggsave(filename = "UMAP_Immune_cells_removed.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```
```{r, fig.width=12}
FeaturePlot(sub_Estrus, reduction = "umap", features = c('Ovgp1', 'Foxj1', 'Fxyd4', 'Cxcl17', 'Myh11', 'Cd52', 'Twist2', 'Pecam1'))
ggsave(filename = "UMAP_Immune_cells_removed1.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```
```{r, fig.width=12}
FeaturePlot(sub_Estrus, reduction = "umap", features = c('Pdgfra', 'Ephx2', 'Dcn', 'Krt19', 'Epcam', 'Vim','Mki67'))
ggsave(filename = "UMAP_Immune_cells_removed2.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```
```{r}
sub_Estrus
```
* After removing immune cells, the dataset is left with:
  + 19418 genes
  + 2318 cells

## Re-run analysis after excluding immune cells
```{r}
previous_time <- proc.time()[3]
sub_Estrus <- RunPCA(sub_Estrus, features = VariableFeatures(object = sub_Estrus))
proc.time()[3] - previous_time
```

```{r, fig.width=12}
DimPlot(sub_Estrus, reduction = "pca") + ggtitle(label = 'Immune cells removed')
ggsave(filename = "PCA_Immune_cells_removed.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```

```{r, fig.width=12}
FeaturePlot(sub_Estrus, reduction = "pca", features = c('Esr1', 'Ovgp1', 'Foxj1', 'Fxyd4', 'Ephx2', 'Dcn'))
ggsave(filename = "PCA_Immune_cells_removed1.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```

```{r, fig.width=12}
FeaturePlot(sub_Estrus, reduction = "pca", features = c('Fos', 'Kcne3', 'Serpina1e', 'Crabp2', 'Pgr', 'Vim'))
ggsave(filename = "PCA_Immune_cells_removed2.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```
```{r}
previous_time <- proc.time()[3]
sub_Estrus_new <- FindNeighbors(sub_Estrus, dims = 1:22)
proc.time()[3] - previous_time
```

```{r}
previous_time <- proc.time()[3]
sub_Estrus_new <- FindClusters(sub_Estrus_new, resolution = 0.04, algorithm = 4)
proc.time()[3] - previous_time
```

```{r}
previous_time <- proc.time()[3]
sub_Estrus_new <- RunUMAP(sub_Estrus_new, dim = 1:22)
proc.time()[3] - previous_time
```

```{r, fig.width=10}
DimPlot(sub_Estrus_new, reduction = "umap") + ggtitle(label = 'Rescale UMAP after removing Immune cells')
ggsave(filename = "UMAP_Rescale.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```
```{r, fig.width=12}
FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Ovgp1', 'Foxj1', 'Crabp2', 'Myh11', 'Wt1', 'S100g', 'Sprr2f',
                  'Fxyd4'))
ggsave(filename = "UMAP_Rescale1.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```

```{r, fig.width=12}
FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Dcn', 'Pax8', 'Pax2', 'Twist2', 'Igf1', 'Serpina1e', 'Pdgfra', 'Krt19', 'Mki67'))
ggsave(filename = "UMAP_Rescale2.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```
## Marker genes
```{r}
marker_genes = c('Epcam', 'Krt15','Krt8','Krt19','Vim','Pdgfra','Twist2', 'Sprr2f', 'Fxyd4', 'Igfbp6',
                 'Acta2', 'Myh11','Foxj1','Ovgp1','Wt1','Serpina1e','S100g','Pecam1')
```

## UMAP for marker genes
```{r, fig.width=12}
FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Epcam', 'Krt15','Krt8','Krt19','Vim','Pdgfra','Twist2', 'Sprr2f', 'Fxyd4'))
ggsave(filename = "UMAP_for_Marker_genes1.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```

```{r, fig.width=12}
FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Igfbp6',
                                                             'Acta2', 'Myh11','Foxj1','Ovgp1','Wt1','Serpina1e','S100g','Pecam1'))
ggsave(filename = "UMAP_for_Marker_genes2.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```

## Rename clusters
```{r}
Cluster.names <- c('1 Ciliated_InfAmp',
                   '2 Secretory_InfAmp',
                   '3 Epithelial_subset',
                   '4 Fibroblast Pdgfra+',
                   '5 Secretory_IsthUTJ',
                   '6 Fibroblast Pdgfra-',
                   '7 Muscle')
names(Cluster.names) <- levels(sub_Estrus_new)
sub_Estrus_new <- RenameIdents(sub_Estrus_new, Cluster.names)
DimPlot(sub_Estrus_new, reduction = "umap", pt.size = 0.5) + scale_color_manual(labels = Cluster.names,values=c('green',                                                                                        'red','blue','orange','grey','yellow','pink','purple'))
ggsave(filename = "UMAP_Clusters_names.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```
```{r, fig.width=12}
DotPlot(sub_Estrus_new, features = marker_genes, group.by = sub_Estrus_new@meta.data$ident) +  
  theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1)) +
  scale_y_discrete(labels = Cluster.names)
ggsave(filename = "DotPlot_for_Marker_genes.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```
## Number of cells in each clusters
```{r}
table(sub_Estrus_new@active.ident, sub_Estrus_new@meta.data$orig.ident)
```

## Extract top genes for each cluster
* Default method of finding highly expressed genes for FindAllMarkers is Wilcoxon Rank Sum test
```{r}
previous_time <- proc.time()[3]
sub_Estrus_new_markers <- FindAllMarkers(sub_Estrus_new, only.pos = TRUE, min.pct = 0.25, logfc.threshold = 0.25)
sub_Estrus_new_markers %>%
    group_by(cluster) %>%
    slice_max(n = 20, order_by = avg_log2FC)
proc.time()[3] - previous_time
```
## Heat map for top 10 genes in each cluster
```{r, fig.width=10}
sub_Estrus_new_markers %>%
    group_by(cluster) %>%
    top_n(n = 10, wt = avg_log2FC) -> top10
DoHeatmap(sub_Estrus_new, features = top10$gene) + NoLegend()
ggsave(filename = "HeatMap_Top10_genes.pdf", plot = last_plot(), device = "pdf",
         path = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Figures/")
```
```{r}
saveRDS(sub_Estrus_new, file = "/home/nguyen/FinalWork/R/ManuallyRemoveGenes/Estrus_subset_analyzed.rds")
```

